Journal: PLoS ONE

Article Title: Induction and Enhancement of Cardiac Cell Differentiation from Mouse and Human Induced Pluripotent Stem Cells with Cyclosporin-A

doi: 10.1371/journal.pone.0016734

Figure Lengend Snippet: A. Gross appearance of cardiomyocyte induction by CSA. Six days after the Flk1 + cell culture on OP9 cells (Flk-d6). cTnT staining (brown). Left panel: control. Right panel: CSA treatment. Scale bars = 400 µm. B. Quantitative evaluation of cardiomyocyte induction by fluorescent intensity of cTnT staining. Relative fluorescent intensity is indicated (n = 4, †, p<0.001 vs control). C. Representative action potential of iPSC-derived spontaneously beating cardiomyocytes. D. Sarcomeric organization in TMRM-purified cardiomyocytes at Flk-d8. Immunostaining with anti-sarcomeric α-actinin antibody (red) and DAPI (blue). Right panel shows higher magnification of boxed area. Scale bar = 25 µm. E–H. Double immunostaining of TMRM-purified cardiomyocytes at Flk-d8 for connexin43 (Cx43) (green) and cTnT (orange) (E), Cav3.2 (green) and cTnT (orange) (F), HCN4 (green) and cTnT (orange) (G), Kir2.1 (green) and cTnT (orange) (H). Nuclei are visualized with DAPI. Scale bars = 25 µm. I. FACS analysis for cardiac progenitor induction from mouse iPSCs by CSA. X axis: CXCR4. Y axis: Flk1. Percentages of FCV cardiac progenitor cells (double positive population; red boxes) in total Flk1 + cell progenies are indicated.

Article Snippet: Anti-Kir2.1 (1∶200) and anti-connexin 43 (1∶200) antibodies were from Alomone (Israel) and Invitrogen (Carlsbad, CA), respectively.

Techniques: Cell Culture, Staining, Derivative Assay, Purification, Immunostaining, Double Immunostaining

Journal: PLoS ONE

Article Title: Epidermal Growth Factor Receptor Down-Regulation Triggers Human Myoblast Differentiation

doi: 10.1371/journal.pone.0071770

Figure Lengend Snippet: Myoblasts were transfected with either control siRNA or siEGFR. A . Percentages of transfected myoblasts with functional Kir2.1 channels, 48 hours post-transfection. B. Current densities of the total population of myoblasts (including myoblasts with no current, i.e. <5 pA). C. Current-voltage relationships of a myoblast transfected with siEGFR. Voltage-steps were to −40, −60, −80, −100, −120 and −140 mV from a holding potential at −60 mV. The inset shows a control myoblast with no Kir2.1 current, a typical Kir2.1 current recorded from a myoblast, 48 h after transfection with siEGFR. Addition of 500 µM Ba 2+ inhibited this current. D. Myoblasts were first transfected with control siRNA or siEGFR, and 24 h later with a plasmid coding for GFP-Kir2.1. One day after, immunoprecipitation of GFP was performed. Immunoblots reveal Kir2.1 and phospho-tyrosine (P-Tyr). E . Cytoplasmic Ca 2+ was assessed with Fura-2-AM on proliferating myoblasts, 2 days after siRNA transfection. Intracellular Ca 2+ stores were depleted with 10 µM thapsigargin (Tg) in a medium containing 250 nM Ca 2+ . Then 1.8 mM Ca 2+ was subsequently added to reveal SOCE. The first part of the experiment was performed with a medium containing 30 mM KCl in order to clamp cells at around −40 mV. The second part was performed with a medium containing 5 mM KCl allowing cells to hyperpolarize. F . Quantification of peak SOCE (n = 6; * p<0.05).

Article Snippet: Blots were incubated with the primary antibodies diluted in 5% non-fat milk or 5% BSA TBST as follows: rabbit monoclonal antibody against EGFR (1∶1500; D38B1 XP®, or 1∶1000; 15F8, Cell Signaling), rabbit polyclonal antibody against MEF2 (1∶3000; sc-313, Santa Cruz Biotechnology), mouse polyclonal antibody against Myogenin (1∶3000; clone F5D, BD Biosciences), mouse monoclonal antibody against α-Tubulin (1∶10000; clone DM1A, Sigma), rabbit polyclonal antibody against Kir2.1 (1∶200; Alomone), mouse monoclonal against phospho-tyrosine (1∶3000; P3300, Sigma) and rabbit monoclonal antibody against phospho-p42/44 MAPK (Erk1/2; Thr2027Tyr204; 1∶1500; D13.14.4E XP®, Cell Signaling).

Techniques: Transfection, Functional Assay, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: Channels

Article Title: Decreased inward rectifier potassium current I K1 in dystrophin-deficient ventricular cardiomyocytes

doi: 10.1080/19336950.2016.1228498

Figure Lengend Snippet: I K1 in wt and dystrophic ventricular cardiomyocytes. (A) Typical original potassium current traces of a wt, mdx, and mdx-utr cardiomyocyte elicited from a holding potential of −100 mV by 500-ms steps to various voltages (the pulse protocol is shown in the inset of Fig. 1B ). The dashed line indicates the zero current level. (B) Current density-voltage relationships derived from a series of experiments as shown in Fig. 1A (n = 38 for wt, 36 for mdx, and 13 for mdx-utr, respectively). The current levels at the end of the test pulse were evaluated and plotted against the applied voltages. The lines through the data points represent fits with the function: y = Y0/(1 + exp((x-V05)/k)). (C) Current density values at −100 mV (means ± SEM) are compared between wt and dystrophic (mdx and mdx-utr) cardiomyocytes. *** indicates that ANOVA revealed a highly significant difference between the tested groups (p < 0.001). (D) Current density values at −100 mV of wt and dystrophic cardiomyocytes from all experiments on female (♀) mice (n = 14 for wt, 15 for mdx, and 13 for mdx-utr). ** p < 0.01, ANOVA. E : Current density values at −100 mV of wt and mdx cardiomyocytes from all experiments on male (♂) mice (n = 24 for wt and 21 for mdx). * p < 0.05 (p = 0.011), Student's t-test.

Article Snippet: Thereafter, the cells were incubated with Anti-Kir2.1 antibody (APC-026, Alomone Labs; 1:500 in PBS) at 4°C overnight.

Techniques: Derivative Assay

Journal: Channels

Article Title: Decreased inward rectifier potassium current I K1 in dystrophin-deficient ventricular cardiomyocytes

doi: 10.1080/19336950.2016.1228498

Figure Lengend Snippet: Kir2.1 protein exparession and localization in wt and dystrophic ventricular cardiomyocytes. (A) Representative western blot experiment of membrane lysates from adult wt and dystrophic (mdx and mdx-utr) ventricular tissues stained for Kir2.1 and the β-subunit of a G s protein (AS7). The latter was used as loading control. (B) Relative band intensities of Kir2.1 normalized to the respective band intensities of the loading control plotted as means ± SEM for wt (n = 5) and dystrophic (mdx, n = 6; mdx-utr, n = 2) animals. A Student's t-test did not reveal a significant difference between wt and mdx (p = 0.11; n.s., not significant). (C) Typical examples of Kir2.1 immunostainings of an isolated wt (left) and mdx (right) cardiomyocyte.

Article Snippet: Thereafter, the cells were incubated with Anti-Kir2.1 antibody (APC-026, Alomone Labs; 1:500 in PBS) at 4°C overnight.

Techniques: Western Blot, Staining, Isolation

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Genetic ablation of smooth muscle K IR 2.1 is inconsequential to the function of mouse cerebral arteries

doi: 10.1177/0271678X221093432

Figure Lengend Snippet: Genetic ablation of K IR 2.1 does not eliminate inward K + currents in cerebral arterial smooth muscle cells (SMCs). Whole-cell patch clamp electrophysiology was used to measure K IR current with voltage ramps from −100 to +20 mV in the absence and presence of Ba 2+ in 60 mM K + . (a,b) Representative recordings of whole-cell and Ba 2+ -subtracted K IR currents in myocytes isolated from SMC K IR 2.1 −/− mice and non-induced Cre SMC controls. (c) Summary data compare peak inward current at −100 mV between groups ( n = 9 SMCs from 6 mice in control group and n = 9 SMCs from 8 mice in knockout group; nested t -test).

Article Snippet: Rabbit polyclonal primary antibodies against K IR 2.1 (APC-026, 1:200; Alomone) and K IR 2.2 (APC-042, 1:200; Alomone) were diluted in quench solution and applied to cells for overnight incubation (4 °C).

Techniques: Patch Clamp, Isolation, Knock-Out

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Genetic ablation of smooth muscle K IR 2.1 is inconsequential to the function of mouse cerebral arteries

doi: 10.1177/0271678X221093432

Figure Lengend Snippet: K I R 2.1 is negligibly expressed in cerebral vascular smooth muscle cells (SMCs) while K IR 2.2 is highly expressed at the cell membrane. Tamoxifen-induced K IR 2.1 knockout significantly reduced subunit expression but levels remained detectable by immunofluorescence. (a) Fluorescent anti-K IR 2.1 (green) exhibited a faint labeling pattern in cerebral arterial myocytes from SMC K IR 2.1 −/− and control mice with nuclei stained with DAPI (blue). (b) Summary of data compares fluorescence intensity (background subtracted) of K IR 2.1 signal between groups ( n = 10 cells from 5 animals in control group and n = 9 cells from 5 animals in knockout group; unpaired t -test). (c) Immunofluorescence labeling of SMCs for K IR 2.2. (d) Summary of data compares background-subtracted fluorescence intensity of K IR 2.2 signal between groups ( n = 8 cells pooled from 4 animals/group; unpaired t -test). Two cells were analyzed per animal with background signal subtracted using 2° antibody control.

Article Snippet: Rabbit polyclonal primary antibodies against K IR 2.1 (APC-026, 1:200; Alomone) and K IR 2.2 (APC-042, 1:200; Alomone) were diluted in quench solution and applied to cells for overnight incubation (4 °C).

Techniques: Knock-Out, Expressing, Immunofluorescence, Labeling, Staining, Fluorescence

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Genetic ablation of smooth muscle K IR 2.1 is inconsequential to the function of mouse cerebral arteries

doi: 10.1177/0271678X221093432

Figure Lengend Snippet: K IR 2.2 protein expression is unaltered in smooth muscle cell (SMC) K IR 2.1 −/− cerebral arteries. (a) Western blot of intact cerebral arteries confirmed that protein levels of K IR 2.2 are not impacted by deletion of SMC K IR 2.1. (b) Summary of data compares K IR 2.2 protein levels between control and knockout mice (normalized to actin; n = 6 mice; unpaired t -test).

Article Snippet: Rabbit polyclonal primary antibodies against K IR 2.1 (APC-026, 1:200; Alomone) and K IR 2.2 (APC-042, 1:200; Alomone) were diluted in quench solution and applied to cells for overnight incubation (4 °C).

Techniques: Expressing, Western Blot, Knock-Out

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Genetic ablation of smooth muscle K IR 2.1 is inconsequential to the function of mouse cerebral arteries

doi: 10.1177/0271678X221093432

Figure Lengend Snippet: K IR 2.1 and K IR 2.2 subunits are inversely expressed in cerebral endothelial and vascular smooth muscle cells. (a) K IR 2.1 and (b) K IR 2.2 subunit expression is shown as average cellular transcript counts per cell, as determined by single-cell RNA sequencing of the mouse brain vasculature. Data highlight differences in the dominant subunit between cell types. Abbreviations: PC – pericytes; SMC – smooth muscle cells; EC – endothelial cells; v – venous; c – capillary; a – arterial; aa – arteriolar. Figures provided by http://betsholtzlab.org/VascularSingleCells/database.html . 33,34

Article Snippet: Rabbit polyclonal primary antibodies against K IR 2.1 (APC-026, 1:200; Alomone) and K IR 2.2 (APC-042, 1:200; Alomone) were diluted in quench solution and applied to cells for overnight incubation (4 °C).

Techniques: Expressing, RNA Sequencing Assay

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Genetic ablation of smooth muscle K IR 2.1 is inconsequential to the function of mouse cerebral arteries

doi: 10.1177/0271678X221093432

Figure Lengend Snippet: Myogenic responses and K + -induced dilation are intact in cerebral arteries of smooth muscle cell (SMC) K IR 2.1 −/− mice. Cerebral arteries from control and SMC K IR 2.1 −/− mice were cannulated and intravascular pressure was elevated stepwise while vasomotor responses were measured. (a) Representative diameter traces from endothelium-denuded vessels of control and SMC K IR 2.1 −/− mice show the effect of increasing pressure on myogenic tone. (b) Summary of data highlights limited impact of smooth muscle K IR 2.1 knockout on myogenic tone development. Paired t -test was performed for 0 vs. 100 μM Ba 2+ treatment ( n = 10 vessels in the control group and n = 11 vessels in the SMC K IR 2.1 −/− group with 1 vessel/mouse; *P < 0.05). (c) K + -induced dilation, elicited by increasing extracellular K + from 5 mM to 10 mM before and after treatment with 100-μM Ba 2+ , was intact in the knockout group ( n = 6 vessels in the control group and SMC K IR 2.1 −/− group; one-way ANOVA with Sidak’s multiple comparisons test). (d) K + -induced dilation (5 mM K + to 10 mM K + ) was abrogated with exposure to low concentrations of Ba 2+ , implicating K IR 2.2 as the mediator of this response based on its Ba 2+ sensitivity profile ( n = 7 vessels from control mice; repeated measures one-way ANOVA with Sidak’s multiple comparisons test). Myogenic tone (%) was calculated as: [(passive diameter – active diameter)/(passive diameter – minimal diameter)] × 100 at each pressure step. K + -induced dilation (%) was calculated as difference between diameter at 10 mM [K + ] and 5 mM [K + ] divided by the dilatory range (passive diameter – minimal diameter).

Article Snippet: Rabbit polyclonal primary antibodies against K IR 2.1 (APC-026, 1:200; Alomone) and K IR 2.2 (APC-042, 1:200; Alomone) were diluted in quench solution and applied to cells for overnight incubation (4 °C).

Techniques: Knock-Out

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Genetic ablation of smooth muscle K IR 2.1 is inconsequential to the function of mouse cerebral arteries

doi: 10.1177/0271678X221093432

Figure Lengend Snippet: Region-specific brain perfusion is not altered in smooth muscle cell (SMC) K IR 2.1 −/− mice at rest and with increased systemic blood pressure. (a) Representative arterial spin-labeled MR brain perfusion maps. Scans were done in a posterior-to-anterior direction and the volume of brain scanned was divided into 5 coronal slices. Resting cerebral blood flow was measured in control and SMC K IR 2.1 −/− mice. Scans were repeated after blood pressure challenge with an intraperitoneal phenylephrine injection. Figure shows slices from 2 regions of the brain (red boxes) spanning cerebral nuclei, hippocampus, thalamus, and hypothalamus. (b) Baseline perfusion in several major brain structures was not significantly different between control and tamoxifen-induced mice. The blood pressure challenge caused a modest but significant rise in cerebral blood flow to a similar extent in control and SMC K IR 2.1 −/− animals. Unpaired t -test was performed for control ( n = 7 mice) vs. SMC K IR 2.1 −/− ( n = 11 mice) comparison; paired t -test was performed for baseline vs. phenylephrine-treatment. *P < 0.05 compared to baseline control.

Article Snippet: Rabbit polyclonal primary antibodies against K IR 2.1 (APC-026, 1:200; Alomone) and K IR 2.2 (APC-042, 1:200; Alomone) were diluted in quench solution and applied to cells for overnight incubation (4 °C).

Techniques: Labeling, Injection